PROJECT SUMMARY/ABSTRACT Numerical chromosomal instability (CIN) is a hallmark of cancer and it results from errors in chromosome seg- regation during mitosis. The role of CIN in tumor evolution is poorly understood and specifically how it supports processes such as metastasis and immune evasion remains unknown. In addition to fueling genomic hetero- geneity, my research has recently demonstrated that CIN also promotes chronic inflammation through the generation of cytosolic DNA. This, in turn, is sensed by the cGAS-STING cytosolic DNA sensing machinery, which, under normal conditions, is activated in response to viral infection. Strikingly, cancer cells co-opt this chronic inflammatory response to spread to distant organs. In this proposal, I will test the central hypothesis that inflammation downstream of CIN represents an attractive therapeutic vulnerability that can be harnessed to selectively target chromosomally unstable tumors. I will examine whether restoration of cell-autonomous (Aim 1) or non-cell autonomous (Aim 2) responses to inflammation can be used to eliminate otherwise ag- gressive and chromosomally unstable tumor cells. Under Aim 1, I will test whether restoring normal cellular responses to cytosolic DNA could selectively target tumor cells that are replete with cytosolic DNA. I will dis- sect regulatory pathways upstream of the cGAS-STING axis with the goal of determining how cancer cells avoid what is an otherwise lethal anti-viral response and type I interferon response. Specifically, I will investi- gate whether MST1 prevents interferon signaling through inhibitory phosphorylation of Interferon Regulatory Factor 3 (IRF3). Such a possibility would invoke the potential use of MST1 inhibitors as a strategy to target chromosomally unstable tumors. Furthermore, I will examine whether tumor cells rely on autocrine M-CSF sig- naling to reinforce survival and migration programs that are particularly critical in the presence of cytosolic DNA. Under Aim 2, I will test the hypothesis that cGAS activation in chromosomally unstable tumor cells pro- motes innate immune activation and that potentiating STING signaling in the tumor microenvironment would restore anti-tumor immunity. I will test whether this can be achieved through the inhibition of ENPP1, an extra- cellular enzyme involved in the hydrolysis of the STING activator, cGAMP. I will also explore whether the use of a Plk4 inhibitor, known to promote chromosome segregation errors, can be used to augment the anti-tumor immune effect of immune therapies and ionizing radiation. Finally, single-cell sequencing and cytokine profiling will be adapted to identify additional mechanisms by which CIN shapes the immune microenvironment allowing tumor cells to thrive despite the presence of a robust immune infiltrate. Identification of these adaptive mecha- nisms would enable the development of therapies that augment the systemic immune effect of DNA damaging therapies such as ionizing radiation. In summary, this work will lead to the development of novel strategies that exploit CIN, an otherwise deadly property, for the purpose of a therapeutic benefit. !